Electrophoresis is a widely used procedure for separating charged molecules, such as proteins, peptides, amino acids, nucleic acids and other macromolecules based upon the mobilities of the molecules in an electric field.
Electrophoretic separations are nearly always carried out in gels as supporting medium, the latter serving as molecular sieves that enhance separation. Polyacrylamide gel is frequently used due to its high chemical and mechanical stability. Polyacrylamide gel electrophoresis is often referred to as PAGE.
Proteins and peptides are usually denatured and treated with sodium dodecyl sulphate (SDS), an anionic detergent, prior to the electrophoresis, so-called SDS electrophoresis. Most peptides bind SDS in a constant weight ratio which give them essentially identical charge densities, and their migration velocities in a polyacrylamide gel of suitable porosity are as a consequence related to their molecular weights. Thus, prior to application to the polyacrylamide gel in SDS-electrophoresis, samples are denatured by heating in the presence of excess SDS and a thiol reagent, usually 2-mercaptoethanol or dithiothreitol.
In the earliest publications describing SDS-electrophoresis, continuous phosphate buffers were used (Shapiro, A. L., Vinuela, E., and Maizel, J. V., (1967) Biochem. Biophys. Res. Commun. 28, 815; Weber, K., and Osborne, M., (1969) J. Biol. Chem. 244, 4406). In a continuous buffer system, the same buffer of a chosen pH and ionic strength is used in the gel and in the electrode chambers.
Today, however, the technique is almost solely used with the discontinuous buffer system described in Laemmli, U. K. (1970) Nature (London) 277, 680. In discontinuous systems, the pH of the separation gel normally differs from that of the buffer. The sharpness of the sample zones may be improved by providing a "stacking gel" with higher porosity (lower polymer concentration) and significantly different pH, on top of the separation gel. In the original version used by Laemmli, the high porosity stacking gel contained 0.125 M Tris-Cl buffer, pH 6.8, while the low porosity separation gel contained 0.375 M Tris-Cl buffer, pH 8.8. The electrode reservoirs contained 0.025 M Tris, 0.192 M glycine (pH.apprxeq.8.3) and 1 gram SDS/liter. When voltage is applied to this system, glycine starts to enter the stacking gel and a sharp boundary (front) will form between a leading chloride-containing zone and a trailing glycine-containing zone. Due to the low ionisation of glycine in the latter zone (resulting pH 8.9), the passage of the front will be accompanied by a drastic increase of voltage, and peptides applied to the top of the stacking gel will concentrate in a narrow very sharp zone behind the front. When the trailing zone enters the separation gel, the pH and the mobility of glycine will increase and simultaneously protein mobilities will decrease due to the lower porosity of the separation gel. With a correctly chosen porosity of the separation gel, peptides of interest will acquire a lower mobility than glycine, "destack" and move in the separation gel with relative velocities mainly determined by their size. In today's practice it is not uncommon to use simplified versions of Laemmli's buffer system. The separation gel buffer may be used as buffer also in the stacking gel, which for a majority of the applications suffice to give sharply stacked peptide zones behind the front. In many cases it is also possible to completely omit the stacking gel and rely solely on the zone sharpening which results within the sample and in connection with sample entrance into the gel. Discussions on the use of Laemmli's discontinuous buffer system can be found in textbooks on electrophoretic techniques (Andrews, A. T., Electrophoresis, Claredon Press, Oxford 1987; Dunn, M. J., Gel electrophoresis: Proteins, Bios Scientific Publisher, Oxford 1993) and detailed experimental protocols are found for example in Ausubel, F. M., et al, Current Protocols in Molecular Biology, Vol. 2, Chapter 10.2, John Wiley & Sons, New York, 1993.
The gels used for stacking and separation, respectively, are normally produced from acrylamide with N,N'-methylene-bisacrylamide (BIS) as a cross-linker, where the BIS concentration normally chosen falls in the range of 2 to 5% by weight of the total monomer concentration. The total monomer concentration used in practice varies between 4 and 20 grams pro 100 ml of gel solution. A number of alternative acrylamide derivatives, such as N,N-dimethyl acrylamide, N-tris(hydroxymethyl)-methylacrylamide and N-hydroxyalkoxyalkyl acrylamide have been suggested to be used instead of acrylamide and there also exist alternative water soluble divinyl compounds, such as N,N'-diallylditartardiamide or N,N'-diacryloylpiperazine, which can be used instead of BIS (U.S. Pat. No. 7,159,847). The polymerization of the gels is normally accomplished with a catalyst system comprised of ammonium persulphate and N,N,N',N'-tetramethylene ethylenediamine (TEMED). Other types of redox initiators for radical polymerization can also be used as well as UV-initiators.
The amide groups in polyacrylamide and most acrylamide derivatives are slowly hydrolysed at the pH of 8.8 used in Laemmli's gel buffer recipe. The hydrolysis proceeds also at low temperature and the carboxylic groups formed are incorporated in the polymer and will generate electro-endosmosis in connection with electrophoresis. The visible effect after 2-3 weeks storage of a gel in a refrigerator prior to electrophoresis is a marked decrease of the distance travelled by the proteins. 3-4 months storage in a refrigerator results in a complete deterioration of the protein separation. Laemmli's buffer system gives excellent results as long as the gels are used shortly after preparation, but it will not give the user reproducible, high quality results when ready-made gels are utilized. The speed of hydrolysis decreases with the hydroxyl ion concentration and in a discontinuous buffer system suitable to combine with ready-made gels for SDS-electrophoresis, the original pH of the gel should not be allowed to exceed 8, and preferably the pH value of the gel should be .ltoreq.7.5.
There exist theoretical models describing the concentration, pH and conductivity changes appearing at the moving boundaries generated in electrophoresis with discontinuous buffer systems (Jovin, T. M., 1973, Biochemistry 12, 871-898; Everaets, F. M., Beckers, J. L., and Verheggen, T. P. E. M., Isotachophoresis, Elsevier, Amsterdam, 1976). Computer programs are also available which allow the calculation of the conditions in front of and behind the moving boundary from electrophoretic mobilities and pK values of the compounds constituting the discontinuous buffer system. From information available, a number of discontinuous buffer systems can be defined were the initial pH in the separation gel is well below 8 at the same time as the mobility of leading ion and conductivity in front of the boundary combined with the mobility of trailing ion and conductivity behind the boundary define conditions which concentrate peptides to a sharp, narrow zone in the stacking gel, and in the separation gel give R.sub.f -values (R.sub.f =distance travelled by peptide divided by the distance travelled by moving boundary) similar to those given by Laemmli's buffer system (Chrambach, A., and Jovin, T. M., 1983, Electrophoresis 4, 190-204).
One such system utilizing 0.112 M Tris-acetate, pH.apprxeq.6.5, as gel buffer and tricine as trailing ion is used in commercial gels for SDS-electrophoresis. These gels are also utilized for native protein electrophoresis in which case alanine is used as the trailing ion (Phast System Separation Technique File no 110, Pharmacia Biotech AB, Uppsala, Sweden).
U.S. Pat. No. 4,481,094 suggests the use of 2-amino-2-methyl-1,3-propane-diol at pH 6.4-7.3 as gel buffer combined with taurine as trailing ion.
DE-A-41 27 546 describes a buffer system with Tris-formate, pH 7.0-8.5, as gel buffer and taurine as trailing ion, while EP-A-0 509 388 discloses an almost identical buffer system solely differing in that formate is replaced by an anion from the group of acetate, chloride, sulphate and phosphate, and that the useful pH range for the gel buffer is given as 7.0-8.0.
EP-A-0 566 784 describes a buffer system where the gel buffer contains an acid with a pK less than 5, an amine having a pK value between 8 and 8.5, and an ampholyte having a pK.sub.2 between 7 and 11 where the ratio of amine to acid should be between 1:0.6 and 1:1 parts by gram equivalent and the ratio of acid to ampholyte between 1:0.5 and 1:4 parts by gram equivalent. Tris is given as the preferred base in the gel buffer as well as in the electrode solutions. Preferred ampholytes in the gel buffer may be selected from the group of glycine, serine, asparagine, .alpha.-alanine, N-tris(hydroxymethyl)methyl-3-aminopropanesulphonic acid, tricine and N-tris-(hydroxymethyl)methyl-2-aminoethanesulphonic acid, while the preferred ampholyte in the cathodic electrode solution is glycine or tricine.
While these known approaches give storage stable polyacrylamide gels for SDS-electrophoresis and, in the Tris-acetate/Tris-tricine or Tris-Cl-glycine/Tris-glycine (EP-A-0 566 784) systems, also R.sub.f -values similar to those given by Laemmli's buffer system, there are still very notable differences between the resulting band patterns. When pure proteins or standard mixtures are run with the Laemmli system, the result is sharp, well defined bands and the number of stained bands normally agrees with that expected. When the other described buffer systems are used, a number of proteins give broadened and/or diffuse bands and an appreciable number of proteins give extra bands which are generated in connection with the electrophoretic run.